The pregenomic RNA, serves as a template for reverse transcription and encodes the reverse transcriptase for Hepatitis B virus (HBV) replication, has the energetically flexible secondary and tertiary structures, which may reduce the accessibility of ribozyme. To identify accessible target sites for hammerhead ribozyme, the secondary structure analysis of the full-length HBV pregenomic RNA was analyzed using the newest bioinformatic tools. Screening the accessible sites for ribozyme based on the structures show that the accessible sites located on the encode region of S, C, X genes and almost not on the regulation region or the encapsidation signal(ε). Thereby three hammerhead ribozymes targeted different sites were designed and transfected into cells to evaluate the catalytically inactivation of HBV gene expression. Experimental results validated that the encoding regions of S, C, X genes were the effective cleavage sites for hammerhead ribozyme and the ribozyme targeted X gene was most potential to inhibit HBV RNA accu- mulation.